The Impact of Nasal Carriage on Surgical-Site Infections after Immediate Breast Reconstruction: Risk Factors and Biofilm Formation Potential

Background

In patients with breast cancer, post-mastectomy breast reconstruction is part of the patient’s treatment and is associated with higher patient satisfaction and psychological benefits [1]. The rate of breast reconstruction surgery has increased in recent years, with implants being used in 50.0% of the procedures performed [2]. Despite the undoubted benefits of implant-based reconstruction, the procedure can be complicated by implant infections, which affect up to 15.0% of patients undergoing reconstruction [3–5]. The most common causative microorganism is Staphylococcus aureus, which has been shown to account for up to 49.0% of all isolated bacteria [6–9]. The staphylococcal infections are caused mainly by the patient’s own flora [10], and the primary reservoir for these bacteria is the vestibulum nasi [11]. There is strong evidence that nasal colonization of S. aureus is an independent risk factor for developing a subsequent surgical site infection (SSI) with S. aureus, either methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA) [12–14]. The current literature, however, rarely discusses the problem of S. aureus nasal carriage among patients undergoing implant-based breast cancer reconstructive procedures [15]. It is well documented that S. aureus has the ability to produce biofilm and that the microorganisms residing within the microbial biofilm community are different from those with planktonic form [16]. Implant-associated infections caused by microbial biofilm formation on the implant surface are persistent, difficult to treat, and require a complete implant removal [17]. However, the capacity for biofilm formation of S. aureus strains colonizing breast cancer patients as the virulence factor is not well studied.

Therefore, we aimed to evaluate the association between nasal carriage of strains of S. aureus and the incidence of SSI in 124 patients with 132 post-mastectomy breast reconstructions performed at a single oncology center in Bydgoszcz, Poland, between June 2020 and August 2021. We also assessed the ability of colonizing S. aureus strains to form biofilm.

Material and Methods

ETHICS STATEMENT:

Samples from nasal vestibule mucosa were submitted to the clinical microbiology laboratory as a part of routine clinical care for screening of MSSA and MRSA. This test was performed per standard of care and was reported in the patients’ medical records. This study was retrospective and used the medical records of patients who underwent mastectomy and breast reconstruction. The study was approved by the Bioethics Committee at L. Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland (approval No. 578/2021, date of approval: November 16, 2021).

STUDY POPULATION:

This was a retrospective study of patients who underwent reconstructive procedures between June 2020 and August 2021 at the Oncology Center – Prof. Franciszek Łukaszczyk Memorial Hospital in Bydgoszcz. All patients underwent mastectomy and either 1-stage or 2-stage implant-based breast reconstruction for breast cancer. All patients were screened for S. aureus nasal carriage prior to admission to hospital. The preparation of the patient for surgery proceeded under the standards of nursing practice, according to the World Health Organization guidelines [18]. All patients received parenteral antimicrobial prophylaxis, cephazolin i.v. 2.0 g or 1.0 g, in a single dose (depending on body weight and age). In the case of severe beta-lactam allergy, 1 dose of clindamycin i.v. 0.6 g was administered as an alternative to cephalosporin.

POLYMERASE CHAIN REACTION XPERT MRSA/MSSA NASAL ASSAY:

Nasal colonization with S. aureus (either MSSA or MRSA) was examined in clinical samples collected from the right and left nasal vestibule mucosa. The nasal swabs were taken within the 30 days prior to hospitalization and were processed using the Cepheid Xpert SA Nasal Complete assay (Cepheid, Sunnyvale, CA, USA), according to the manufacturer’s instructions on the GeneXpert Dx system (Cepheid). The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple samples using real-time PCR. The primers and probes in the Xpert SA Nasal Complete Assay detect proprietary sequences of the staphylococcal protein A (spa) gene, the gene for methicillin/oxacillin resistance (mecA), and the staphylococcal cassette chromosome (SCCmec) inserted into the SA chromosomal attB site. The results were provided automatically by the GeneXpert Dx system and were positive for S. aureus if the spa gene was detected, and positive for MRSA if the spa, mecA, and SCCmec genes were all detected. The results of the test were available within less than 2 h after collection.

STANDARD MICROBIOLOGICAL CULTURE, STRAIN IDENTIFICATION, ANTIBIOTIC SUSCEPTIBILITY TESTING:

In the case of a positive PCR Xpert MRSA/MSSA nasal test, standard culture methods for S. aureus were followed. The samples were processed using culture-based methods on Staph/Strep Selective Medium (Oxoid Deutschland GmbH, Wesel, Germany) under aerobic conditions at 35±2°C. After 24 h, suspected isolates of S. aureus were identified using a coagulase test, the commercial Dry Spot Staphytect Plus (Oxoid Limited, Basingstoke, Hampshire, England), and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (IVD MALDI Biotyper Smart System, microflex LT/SH smart, Bruker Daltonik, Bremen, Germany). Culture-negative plates were further incubated and examined after 48 h. The screening swabs taken from patients were also inoculated on Brilliance MRSA 2 agar (Fisher Scientific, Loughborough, Leicestershire, UK) for MRSA detection. All agar plates were read after 18 to 24 h of incubation at 35±2 °C, according to the manufacturer’s instructions. If, after this time, no denim blue colonies were observed, the specimen was considered negative for MRSA and the plates were discarded. Susceptibility to antibiotics was determined by the disk diffusion method on Mueller-Hinton E agar (BioMérieux SA, Marcy l’Etoile, France), as recommended by the European Committee on Antimicrobial Susceptibility Testing [19]. The antibiotics used were cefoxitin (disk content 30 μg) and mupirocin (disk content 200 μg; Oxoid Limited). The results were interpreted in accordance with the European Committee on Antimicrobial Susceptibility Testing breakpoint tables v. 11, valid from January 1, 2021 [20]. S. aureus strain ATCC 29213 was used for quality control.

SSI IDENTIFICATIONS:

SSI were diagnosed if clinical signs of infection occurred within 90 days after the operation and diagnosis was made by a surgeon, according to the European Centre for Disease Prevention and Control for superficial and deep incisional infections [21].

STUDY GROUPS:

All cases of patients with immediate post-mastectomy reconstruction were analyzed. A total of 124 patients with 133 reconstructive surgery procedures for breast cancer were identified. Among them, 115 patients underwent unilateral reconstruction, 3 patients underwent bilateral surgery, and 6 patients underwent 2 reconstructions. One of these reconstructions (patient with 2 reconstructions) was excluded from further analysis because they did not meet the adopted criteria, owing to treatment with mupirocin during hospitalization. The patients were stratified into 2 groups according to occurrence of infection. The differences between the groups were evaluated in relation to the possible risk factors for SSI after the placement: age, body mass index (BMI), length of hospital stay, histological form of cancer, American Society of Anesthesiologists (ASA) grading, type of implant, treatment with adjuvant or neoadjuvant chemotherapy (CHTH,) occurrence of risk factors, and number of risk factors. Moreover, according to the PCR result and mupirocin treatment status, patients were separated as follows: PCR negative for MSSA/MRSA, PCR positive for MSSA/MRSA, PCR positive for MSSA/MRSA with mupirocin treatment, PCR positive for MSSA/MRSA without mupirocin treatment.

COLONIZING STRAINS:

In total, 36 S. aureus isolates were obtained from the analyzed patient cases. Four colonizing isolates were not available (due to not being saved in the laboratory); thus, 32 isolates from 31 individual patients were archived and stored in tryptic soy broth (TSB-T; BioMérieux SA, Marcy, l’Etoile, France) with 20% (vol/vol) glycerol at -70±2°C.

DETERMINATION OF THE BIOFILM FORMATION:

The qualitative determination of the biofilm formation was provided by the colorimetric assay with crystal violet staining, as previously described [22,23]. Briefly, fresh bacterial suspensions were prepared in Luria-Bertani broth (LB, Merck KGaA, Darmstadt, Germany), supplemented with 2% glucose (Merck KGaA) from overnight cultures and adjusted to 106 colony forming units (CFU)/mL. The wells of a sterile 96-well flat-bottomed polystyrene microplate (Kartell S.p.a., Medlab) were filled with 200 μL of the overnight bacterial culture into each well. The negative control wells contained LB broth only. After 48 h of incubation in 35±2°C, the medium was gently removed and the microtiter plate wells were washed 3 times with 150 μL sterile 1% phosphate-buffered saline (PBS; Thermo Fisher Scientific, Loughborough, Leicestershire, UK). The temperature-fixed (10 min at 60°C,) biofilms were stained with 100 μL 0.15% crystal violet (Merck KGaA) and stored for 15 min at room temperature. The unbound crystal violet stain was removed, and the wells were washed gently 3 times with 150 μL 1% PBS. The wells were air-dried for 15 min and the crystal violet in each well was solubilized by adding 200 μL 96% ethyl alcohol (Chempur, Piekary Śląskie, Poland). The absorbance (A) of each well with alcohol extracts was measured in new flat-bottomed microtiter plate at 570 nm using an automated Synergy HTX multi-mode reader (Biotek Instruments, Inc., USA). Results were analyzed as biofilm biomass in negative and positive biofilm productions in comparison to the control without bacteria, and the classes were defined as follows: A isolate ≤A control=non-biofilm producer; A control <A isolate <2×A control=weak producer; 2×A control <A isolate <4×A control=moderate; 4×A control <A isolate=strong-producer. Each experiment was performed 3 times in 3 replications, and means and standard deviations were calculated and the 1-way ANOVA test was performed. In the quantitative determination of biofilm formation, the strains of the S. aureus were grown in LB broth, as in the qualitative methods described. After incubation in the microplate, the supernatants were removed and the biofilms were carefully washed 3 times with 150 μL PBS. The biofilms were resuspended in 150 μL PBS by scraping them from the cavities and pipetting up and down several times. From each cavity, an aliquot of 200 μL of cell suspensions was transferred into a 1.5-mL Eppendorf tube and serially diluted in a logarithmic manner up to 10−5. From selected dilutions, 10 μL were dropped on LB agar plates, and the CFU/mL were determined after incubation at 35±2°C overnight. Each experiment was independently performed 2 times, and the results are expressed as mean and standard deviation.

STATISTICAL ANALYSIS:

Statistical analysis was conducted using Statistica (data analysis software system, TIBCO Software Inc., www.statistica.io, version 13.3), IBM SPSS Statistics 27 (IBM Corp, Armonk, NY, USA) data analysis software, and GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). Between-group differences were assessed using the t test for continuous variables and chi-square test (H0 Wald statistics) for categorical variables. Data are presented as a categorized histogram, mean±standard deviation, or the number of cases and percentages, where appropriate. Univariate logistic regression analysis was applied to assess the influence of continuous and categorical variables on the risk of infection. Time-dependent risk of infection was assessed using the log-rank test and visualized using the Kaplan-Meier model, where infection probability was calculated as 1 minus cumulative survival probability. The 1-way ANOVA test was used to show differences between the amount of biofilm produced by replicated groups from the experiment, and the Mann-Whitney U test was used to compare differences between the crystal violet test and CFU methods. In all statistical analyses, the cut-off value for the probability coefficient was set as a P value ≤0.05.

Results

CHARACTERISTICS OF STUDY GROUPS:

A total of 124 patients who had 132 reconstructions were analyzed. The mean age of patients was 45.41 years (±7.21 SD), and the mean BMI was 25.16 (±3.76 SD). Risk factors were present in 79 of 132 patient cases (59.8%). Twenty-two of 132 the analyzed patient cases (16.7%) had 2 risk factors. Characteristics of the whole group are presented in Table 1.

:

The overall, preoperative nasal S. aureus carriage rate among the analyzed patient cases was 28.8% (38/132). The samples of 2 of 38 patients (5.3%) that tested positive in the Xpert SA Nasal Complete Assay did not grow in culture. All S. aureus strains obtained from cultures (n=36) demonstrated susceptibility to mupirocin for nasal decontamination.

BIOFILM FORMATION ASSAYS:

Qualitative results of the biofilm biomass in absorbance (A) of the strains were presented in the following classes: A ≤0.165=non-biofilm producer; 0.165 <A <0.329=weak producer; 0.329 <A <0.659=moderate; and 0.659 <A=strong producer. A total of 31 (96.9%) clinical S. aureus strains had a strong biofilm formation capacity, and 1 strain (3.1%), number 29, showed moderate ability (A=0.482). The maximal absorbance (A=3.046) was observed for strain 3 (Figure 1). The 1-way ANOVA test showed significant differences in the amount of biofilm produced in replicated groups of the experiment (P<0.0001). In the quantitative in vitro test with log10 CFU/mL, measurements of the average viable cells counts ranged between 6.826 (strain number 17) and 9.435 (strain number 20; Figure 1). For most strains, a 2-log increase in viable cells in the CFU biofilm compared with the control inoculation was observed. In 8 cases (25%), the biofilm mass in the crystal violet was higher than CFU assay, which probably can be associated with aggregation of extracellular substances in the biofilm matrix and not with the number of viable S. aureus cells. The Mann-Whitney test showed statistical significance in the amount of biofilm analyzed by crystal violet test and CFU method (P<0.0001).

SSI WITHIN 90 DAYS AFTER RECONSTRUCTION:

The infection rate following reconstruction procedures was 5.3% (7/132). In all cases, we identified deep incisional SSI. Infections occurred both in patients colonized (n=1) and non-colonized with S. aureus (n=6). Among the patients who were PCR positive for MSSA/MRSA without treatment with mupirocin, one 44-year-old S. aureus carrier, after CHTH treatment, developed an infection with S. aureus 70 days after reconstruction. The colonizing S. aureus strain (strain number 19) had a high ability to form biofilm, confirmed by qualitative and quantitative assays, as presented in Figure 1. The mean time to onset of infection was 64.63 (±16.90) days. There was no significant difference between study groups, except regarding the type of implant. The use of an expander tended to be higher in the group with infections (85.7% vs 14.3%, P=0.0312). Comparisons between groups are shown in Table 1. BMI data are presented as a categorized histogram and are shown in Figure 2. The length of hospital stay in the 2 groups was similar, being 8.8 (±2.9) days for cases without infection vs 8.9 (±3.9) days (P=0.9328) for cases with infections.

RISK FACTOR ANALYSIS FOR IMPLANT-BASED BREAST RECONSTRUCTION INFECTIONS:

Patients treated with CHTH had around a 4-fold higher risk (95% CI 0.75, 21.5; P=0.1049) for developing infection. Using an expander led to an almost 8-fold higher risk (95% CI 0.89, 65.32; P=0.0634), and there was an almost 5-times higher risk when 2 risk factors were present (95% CI 0.43, 57.59; P=0.2012). However, none of the analyzed risk factors had a statistically significant influence on the occurrence of implant-based breast reconstruction infections. The risk of infection in univariate analysis is presented in Table 2. Time-dependent risk of infection for number of risk factors, type of implant, PCR status of MSSA/MRSA, and treatment with CHTH is shown in Figure 3A–3D. The cumulative infection probability was higher for the use of an expander than for a prosthesis (P=0.031), and for treatment with CHTH than without CHTH treatment (P=0.089); however, the differences in relation to CHTH were not statistically significant.

Discussion

STRENGTHS AND LIMITATIONS OF THE STUDY:

The limitations of this work include the relatively small group of patients with infections, the retrospective nature of the work, and the lack of in-depth microbiological analysis determining the causative agents of infection among non S. aureus carriers. Another limitation is the short observation time. Strengths include the new approach to the problem of infections after mastectomy reconstruction, the combination of known risk factors, and the S. aureus colonization status of the patients with ability to form biofilm by colonizing strains.

Conclusions

There was no association between nasal carriage of strains of S. aureus and the incidence of SSI. However, further investigations on a larger group of patients and a longer observation time are needed to investigate this potential risk factor in detail.